N-terminal sequence analysis of polypeptide at the picomole level.
This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.
D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D017879:Isothiocyanates; D008722:Methods; D010455:Peptides / Q000378:metabolism*; D013861:Thiocyanates; D013867:Thiohydantoins; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D000596:Amino Acids; D007202:Indicators and Reagents; D017879:Isothiocyanates; D010455:Peptides; D013861:Thiocyanates; D013867:Thiohydantoins; C005441:phenylisothiocyanate; C022927:DABTH; C012926:4-(N,N-dimethylaminoazobenzene)-4'-isothiocyanate; D004124:p-Dimethylaminoazobenzene